Microbio Lecture 8 Environmental Sensing and Chemotaxis

What are the two component to two component regulatory systems?

Component 1: The Sensor Histidine Protein Kinase

Component 2: Response Regulator

Two Component Regulatory Systems

Important mediators of signal transduction that allows bacteria to detect physical/chemical changes and relay this signal to respond

Sensor Histidine Protein Kinase

\-Membrane associated or cytoplasmic

\-autophosphorylates a conserved histidine residue environmental signal

\-sensors consists of at least two domains:

a. variable response domain (why variable?)

b. conserved transmitter domain (histidine protein kinase); where they get phosphorylated

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\-sensors usually form dimers and exhibit autokinase activity (ATP binds to one subunit and phosphorylates the other in a process known as transphosphorylation)

\-sensor transmits phospho-group from histidine residue to aspartate residue on response regulator

Transphosphorylation

Phosphate group is transferred to the receivor

Response Regulator

\-second part of 2 component regulatory system

\-consists of two domains: receiver domain with aspartate residue and variable regulator

\-different phosphorylated response regulators have different half lives; length of half life determines duration of response to changing environmental conditions

\-many dimerizes after phosphorylation and bind to DNA

What determines length of the duration of a response?

It depends on the half life of phosphorylated response regulators

Do all response regulators bind to DNA?

No not all of them do ex. CheY and CheB

CheY and CheB

two response regulators involved in chemotaxis; they dont bind to DNA at all but modulate activity of other proteins

EnvZ/OmpR System of E. Coli

\-Two Component Regulatory System

\-Has EnvZ sensor kinase and OmpR regulator

1\. Signal binding causes activation of the autokinase resulting in ATP hydrolysis and phosphorylation of the histidine on phosphotransfertase subdomain

2\. Phosphoryl group transferred to aspartyl group (D) on response regulator

If ompF is upregulated by low osmolarity and ompC is upregulated by high osmolarity, which encodes a porin protein with a larger diameter?

OmpF does

Sporulation Response of B. Subtilis

\-sensor kinase sends signal to series of proteins before it reaches to the response regulator where it interacts with DNA

Taxis

Oriented movement of motile organisms

What does taxis require?

motility and sensors

How does the sensor help the taxis?

It helps the bacteria know whether to go forward or which direction

What are taxis distinguished by?

They are distinguished by the basis of stimuli:

\- Chemotaxis (chemical substances)

\- Phototaxi (light)

\- scotophobotaxis (darkness)

Chemotaxis

Prokaryotes have gradient sensing mechanism that allows them to compare concentration of a chemical over short time intervals rather than having to make instantaneous comparison in space

\-mechanism makes it possible for them to make spatial comparisons over many cell lengths rather than a single cell length

Bacterial Flagellum

Has a hook that helps it rotate via the motor switch (Fli proteins)

Fli Proteins

Motor switch for bacterial flagellum

What are some different types of flagella?

Peritrichous, polar, and lophotrichous

What modes of movements is chemotaxis based on?

Going forward or stop and tumble

What happens when bacteria rotates counterclockwise?

Moves forward

What happens when bacteria rotates clockwise?

They stop and tumble which can allow them to switch to a different direction

Random Walks

In absence of a chemical gradient, bacteria move in a random fashion that includes runs and tumbles

\-generates a random movement

Biased Random Walk

\-can’t make conscientious decision to walk somewhere

\-in presence of gradient of attractant/repellant, bacteria bias their random walk to include longer runs and fewer tumbles up/down concentration gradient

What type of flagella does E. coli have?

\-Peritrichous

\-Counterclockwise Rotation → Smooth Swimming

\-Clockwise → Flagellar bundles fly apart resulting in tumbling

Clockwise Movement

Tumbling

Counterclockwise Movement

Smooth swimming

Bacterial Chemotaxis

A set of proteins that has a sensor histidine kinase that gets signals from attractants and then sends signals to response regulator to modulate the chemotaxis by alternating duration of periods of smooth swimming and tumbling

Sensory Proteins

Tar and Tsr proteins; methyl accepting chemotaxis proteins (MCPs) that act as the bacteria’s “nose”

MCP

methyl accepting chemotaxis proteins; acts as the bacteria “nose”

Tar

an MCP that senses aspartate and maltose as well as repellants cobalt and nickel; have multiple binding sites in sensor domains for attractants and repellants (each causes different conformational change)

What are the MCPs in contact with?

They are in contact with CheA and CheW

CheA

a sensor histidine kinase protein that is held by CheW

CheW

Protein that is in contact with MCP and CheA and helps alter rate of autophosphorylation of CheA

What does ligand binding to MCP result in?

Conformational changes that change rate of CheA autophosphorylation with help of CheW

What is the response regulator of CheA?

CheY; CheA transfers the phosphyl group to the aspartate residue of CheY

CheY

Response regulator of CheA; when phosphorylated it interacts with FliM to change rotation of bacteria from CCW to clockwise to cause tumbling

FliM

protein that is component of flagellar motor, responsible for changing direction of the bacteria movement

Does more attractant increase or slow the rate of autophosphorylation?

It slows the rate of autophosphorylation

What does phosphorylated CheA do?

It transfers its phosphate to aspartate residue on response regulator CheY

CheY

a response regulator

What does phosphorylated CheY do?

It interacts with FliM (part of the flagellar motor) to change direction of rotation from CCW to CW (results in tumbling)

What happens when an attractant is added to chemotaxis?

1\. In response to attractant binding to MCP, CheA slows its rate of autophosphorylation

2\. In absence of CheA\~P, CheY remains unphosphorylated and does not interact with FliM; CheZ helps keep CheY dephosphorylated in low levels of CheA\~P

3\. Disrupts CheY-FliM interaction switches flagellar to CCW rotation and continues to do smooth swimming

CheZ

Protein that keeps CheY unphosphorylated in low levels of phosphorylated CheA

What happens when repellant is added to chemotaxis?

1\. Repellant binding by MCP leads to increase in CheA autophosphorylation

2\. CheA\~P transfer phosphate to CheY and stimulates its interaction with FliM

3\. CheY\~P-FliM interaction switches flagellar motor to clockwise movement and initiates tumbling

Adaptation of Chemotaxis

\-Adaptation allows resetting of signal state of MCP’s altho concentration of attraction remain the same

ex. cells bias their random walk up a gradient but can adapt to do random walk in high (but unchanging) conc. of the attractant

Sporulation

Process of bacteria asymmetrically dividing creating a spore that will break out of mother cell

Spore can survive in extreme environmental conditions

Sporulation in B. Subtilis

What does B. Subtilis sporulation involve?

\-Quorum Sensing: Pheromones

\-Cell-cell signaling: -phosphorelay

\-Transcriptional regulation: sigma factors

\-Cell Type Specific Proteolysis - SigmaE and K

B. Subtilis Endopore

\-they are remarkably resistant

\-can survive high temperatures, UV lights, and in organic solvents

What triggers sporulation?

\-Crowding

\-Starvation

\-Lack of options

* kind of a last ditch process that is irreversible once it started and requires a lot of energy

What is sporulation governed by?

By a cascade of transcriptional factors

Sigma F

Sigma F triggers the start of the mother cell tries to engulf the spores

Sigma E

Causes the mother cell to finish engulfing the spores

Sigma G

Triggered by the finishing of the mother cell engulfing the spores

Sends a signal via a transduction pathway to Sigma K in mother cell to process it from an inactive pro-protein into an active transcription factor

Sigma K

Protein in the mother cell

\-gets activated by sigma G transduction

Add additional proteins in between the spore’s layers to make it more resistant to environmental conditions; adds the cortex

Sigma H and Spo0A

\-activated during stationary phase via a two component regulatory system

\-trigger the process of asymmetric cell division

What is the result of sporulation?

An endospore is formed and the mother cell degrades

What phase does sporulation occur?

It occurs during stationary phase

Spo0A

Response regulator activated at the end of a phosphorelay of a two component regulatory system

When does Spo0A and Sigma H get activated?

During the stationary phase; they are activated via two component regulatory system

Quorum Sensing

\-Critical component of sporulation

\-helps sense that the area is crowded and sense if there is presence of food or not

Rap Phosphatases

Proteins that can dephosphorylate Spo0F

\-block transfer of phosphyl group from Spo0F to Spo0A

\-activity dependent on quorum sensing

What deactivates Rap Phosphatase?

Peptide signals

Quorum Sensing in B. Subtilis

cells secrete and respond to a set of small peptides via the ABC transporter Spo0K

Phr and CSF

peptides that repress Raps and promote Spo0A activation

What do you need to initiate sporulation?

Both Spo0A and Sigma H

How is Sigma H activated?

It’s activated in part by Spo0A\~P dependent transcription of the sigH gene

How would you test that Sigma H activation is related to Spo0A transcribing sigH gene?

Test this by creating a mutant that does not produce the sigH gene

Significance of Spo0A and SigmaH in Sporulation

Drive expression of genes responsible for the switch from medial to polar septation

What does Sigma E and Sigma F do in sporulation?

Required for mother cell engulfing the spore

What proteins are important for the engulfment in sporulation process

Sigma E and Sigma F

What is engulfment coupled to in the forespore?

Engulfment is coupled to Sigma G Activation in the forespore